Title: The interplay between RAD51C sub-complexes during replication fork repair Abstract: Alkylating agents are an environmentally relevant class of DNA damaging compounds that can cause replication fork damage and subsequent collapse leading to double-strand breaks (DSBs). One important group of proteins used to repair DSBs includes the RAD51 paralogs. However, the role of the RAD51 paralogs in response to alkylation-induced DNA damage has been understudied in human cells. The RAD51 paralog family consists of five proteins in humans, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. They are known to assemble into sub-complexes with themselves and with other homologous recombination (HR) factors, but how each complex functions, and the DNA substrates they act upon remains undetermined. RAD51C is a member of multiple sub-complexes, and importantly mutations in RAD51C are linked to hereditary breast and ovarian cancers and to Fanconi Anemia. Recently, the RAD51 paralogs have been found to localize to replication forks, but their recruitment to damaged forks, such as those induced by alkylation damage, remains to be investigated. My work will determine the importance of RAD51C in the repair of alkylation-induced DNA damage at replication forks. Using environmentally relevant alkylating agents and the prototypical methylating agent, methylmethane sulfonate, I will measure RAD51C presence at replication forks using the new technology, isolation of proteins on nascent DNA (iPOND). Further, by depleting RAD51C complex members in conditional knockout cell lines, I will use immunofluorescence and iPOND to visualize the consequential changes in RAD51C localization to determine which RAD51C containing sub-complexes are involved in repair of alkylation-damaged forks. Using yeast-two-hybrid analysis to screen cancer-associated RAD51C mutations, I will identify mutants that disrupt individual RAD51C-associated complexes. RAD51C separation-of-function mutations selected for functional analysis will be validated for interaction changes by co- immunoprecipitation and in vitro pull-downs. Using these RAD51C mutations, I will determine at which steps of repair RAD51C-associated complexes regulate repair in response to alkylation damage. These complementary approaches will determine the contribution of RAD51C in repair of alkylation-induced DNA damage and its overall role in preserving genomic stability. Further, this work will aid in our understanding of the significance of environmental factors that contribute to the cancer predisposition of individuals with RAD51 paralog mutations.